Dynamin on Comets
The large GTPase dynamin (Dyn2) has been demonstrated by us and others to interact with several different actin-binding proteins. To define how Dyn2 might participate in actin dynamics in livings cells we have expressed green fluorescent protein (GFP)-tagged Dyn2 in cultured cells and observed labeling of comet-like vesicles and macropinosomes. The comet structures progressed with a constant velocity and were reminiscent of actin comets associated with motile vesicles in cells expressing type I phosphatidylinositol phosphate 5-kinases. Based on these observations we sought to determine whether Dyn2 is an integral component of actin comets.
Cells expressing type I phosphatidylinositol phosphate 5-kinase and Dyn2-GFP revealed a prominent colocalization of Dyn2 and actin in comet structures. Interestingly, comet formation and motility were normal in cells expressing wild-type Dyn2-GFP but altered markedly in Dyn2 mutant-expressing cells. Dyn2K44A-GFP mutant cells displayed a significant reduction in comet number, length, velocity, and efficiency of movement. In contrast, comets in cells expressing Dyn2DPRD-GFP appeared dark and did not incorporate the mutant Dyn2 protein, indicating that the proline-rich domain (PRD) is required for Dyn2 recruitment. Further, these comets were significantly longer and slower than those in control cells. These findings demonstrate a role for Dyn2 in actin-based vesicle motility.
The large GTPase dynamin regulates actin comet formation and movement in living cells
Dyn2-GFP incorporates into endogenous comet-like vesicles and macropinosomes in living cells
(a) Confocal time-lapse imaging of Clone 9 hepatocytes expressing Dyn2-GFP. Dynamic comet-like structures were observed. N, nucleus. (b-e) Higher magnification revealed that these motile structures consisted of a brightly stained head followed by a tail structure (arrows) that extends 180° from the direction of movement. (f) The velocity of the movement was uniform as revealed by the consistent distances between projected frames of the time-lapse images (arrows). (g-k) Confocal time-lapse imaging of Dyn2-GFP in NIH 3T3 fibroblasts revealed large vesicular structures associated with small tails of Dyn2-GFP (arrows and boxes). (h' and k') High magnifications of individual macropinosomes from frames h and k, respectively. Consistent with the formation of macropinosomes, these structures formed from zones of active membrane ruffling at the cell periphery. (Bar, 10 mm.)
Dyn2 is an integral component of actin comets
Cultured rat fibroblasts and Clone 9 hepatocytes showing dynamin incorporated into PIP5KI a-induced actin comets. (a and a') Comets in a rat fibroblast expressing Dyn2aa-GFP and colabeled with cortactin. The Dyn2 labeling is brightest at the head of the comet (arrows) but is prominent also in the tail (Inset, arrows). In contrast, cortactin staining is localized predominantly to the comet tail. RF, rat fibroblast; aCortY, anti-cortactin. (b and b') Comets in a Clone 9 hepatocyte immunolabeled for dynamin ( aDyn2) and actin (rhodamine-phalloidin). Note the Dyn2 enrichment in the head and staining in the tails (arrows and Inset), whereas actin stains the tail only. (a: bar, 3 mm; b: bar, 2 mm.)
Dyn2 regulates comet formation, actin tail length, and comet velocity
For quantitation of comet formation and actin tail length, rat fibroblasts coexpressing Myc-PIP5KIa and wild-type or mutant Dyn2-GFP were fixed and stained for actin (rhodamine-phalloidin). (a) Cells expressing the K44A mutant show on average only 1.2 comets per cell, a 72.1% reduction compared with cells expressing PIP5KIa alone (4.3), and Dyn2DPRD expression shows a reduction in comet formation of 25.6% (3.2 comets) compared with PIP5Ka control. (b) The average comet tail length in cells expressing PIP5KIa and Dyn2 was only 6.5% longer than PIP5KIa control (5.7 mm), whereas those in cells expressing Dyn2K44Awere 40.8% shorter (3.2 mm). In contrast, comet tails in Dyn2DPRD-expressing cells were 16.5% longer (6.2 mm) than control. (c) Particle-tracking analysis showed that Dyn2K44A-GFP and Dyn2DPRD-GFP comets progressed with reduced velocities of 0.09 and 0.11 mm/s, respectively, whereas wild-type Dyn2-GFP comets progressed at 0.14 mm/s. The movies below, also published as supporting information on the PNAS web site, show comparison of the Dyn2 comets.
Dynamin and Cortactin Comets
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